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Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica
Thesis (MSc)--Stellenbosch University, 2015.
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| Format: | Thesis |
| Language: | en_ZA |
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Stellenbosch : Stellenbosch University
2015
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| _version_ | 1867613982007754752 |
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| access_status_str | Open Access |
| author | Govender, Seshni |
| author2 | Van Zyl, Willem Heber |
| author_browse | Govender, Seshni Van Zyl, Willem Heber |
| author_facet | Van Zyl, Willem Heber Govender, Seshni |
| author_sort | Govender, Seshni |
| collection | Thesis |
| dc_rights_str_mv | Stellenbosch University |
| description | Thesis (MSc)--Stellenbosch University, 2015. |
| format | Thesis |
| id | oai:scholar.sun.ac.za:10019.1/96628 |
| institution | Stellenbosch University (South Africa) |
| language | en_ZA |
| last_indexed | 2026-06-10T12:44:46.833Z |
| license_str | Other — see source repository |
| provenance_str_mv | Harvested via OAI-PMH from SUNScholar — Stellenbosch University Repository |
| publishDate | 2015 |
| publishDateRange | 2015 |
| publishDateSort | 2015 |
| publisher | Stellenbosch : Stellenbosch University |
| publisherStr | Stellenbosch : Stellenbosch University |
| record_format | dspace |
| source_str | SUNScholar — Stellenbosch University Repository |
| spelling | oai:scholar.sun.ac.za:10019.1/96628 Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica Govender, Seshni Van Zyl, Willem Heber Van Zyl, P. J. Crampton, M. Stellenbosch University. Faculty of Science. Dept. of Microbiology. Yarrowia Lipolytica Exenatide Lip2 protein production -- Economic aspects Hp42 promoter Yeast -- Fermentation UCTD Thesis (MSc)--Stellenbosch University, 2015. ENGLISH SUMMARY: Exenatide (Byetta®) is a type 2 diabetic drug which decreases the blood glucose level. Treatment using this drug is expensive due to its costly production by chemical synthesis therefore it is not an affordable drug of choice. A potential cost effective alternate for the production of exenatide is the use of recombinant production technology. Yarrowia lipolytica, a dimorphic yeast, was genetically engineered to produce the exenatide peptide (Exe, 39 amino acids) as a fusion to Lip2 (lipase) protein under the regulation of the hp4d promoter by the CSIR. The regulation of the promoter has, until recently, not been elucidated and is currently reported to be growth phase dependent. In order to optimise the conditions for the production of the fused Lip2:Exe peptide precursor, the regulation of the promoter needed to be understood. In this study, the regulation of the hp4d promoter was established and a fed-batch fermentation strategy for the production of the fused Lip2:Exe precursor was developed. A Y. lipolytica strain (YlEx-gly) producing the Lip2:Exe peptide was cell-banked (to ensure stability of the production organism and repeatability of inoculation of fermenters) and the cell-bank was validated for production of the fused peptide. A transcript profile of the recombinant strain harbouring an expression vector encoding the Lip2:Exe under control of the hp4d promoter was determined using an optimised mRNA sandwich hybridisation methodology. Batch fermentation (1.2 l) was used to monitor production profiles during growth of Y. lipolytica followed by continuous fermentation (1 l) to determine the effect of growth rate on the transcription levels of the product under regulation by the hp4d promoter. The synthetic hp4d promoter was found to be growth rate dependent which was confirmed by quantifying the amount of total protein produced during fed-batch fermentations (10 l) at different growth rates. A 60 % increase in production yields was achieved by using the optimised growth rate of 0.02 h-1. This validated that the hp4d is growth rate dependent and not growth phase dependent as reported in literature. A strategy for the recombinant production of pharmaceutical peptides and proteins, under regulation of the hp4d promoter, using Y. lipolytica as a host, was therefore established. This research has paved the way for recombinant production of proteins at a lower cost therefore impacting on the health and economy of South Africa, by providing the public with potentially cheaper, affordable pharmaceutical drugs due to an alternative production strategy. AFRIKAANS OPSOMMING: Exenatide (Byetta®) is a middle wat vir die behandeling van tipe 2 diabete gebruik word deur die bloed glucose konsentrasiete verlaag. Hierdie behandeling is egter baie duur aangesien dit chemiesvervaardig word en dus nie bekostigbaar vir alle pasiente is nie. ‘n Bekostigbare alternatiewe vervaardigingsmetode is die gebruik van rekombinante produksie. Yarrowia lipolytica is ‘n dimorfiese gis wat geneties gemanipuleer is om exenatide (Exe, amino sure) as ‘n fusieproduksaam met die Lip2 (lipase) proteïen onder regulering van die hp4d promoterte produseer. Die regulering van die promoter is onbekend endit word aanvaar dat uitdrukking afhanklik is van die groeifase. Om produksie van die Lip2:Exe peptiedvoorloper te optimiseer is dit belangrik om die regulering van die promoter te verstaan. In die studie is die reguleering van die hp4d promoter bepaal en ‘n voerstrategie vir die produksie van die Lip2:Exe peptiedvoorloperin voer-lotfermentasie ontwikkel. ‘nY. lipolytica kloon (YlEx-gly) wat die Lip2:Exe peptiedvoorloper produseer is in ‘n selbank gepreserveer om die stabiliteit van die produksie organisme en herhaalbaarheid van die inokulasie van die fermenteerders te verseker) en die selbank is getoets vir produksie van die fusiepeptied. ‘n Transkripsie profiel van ‘n positiewe kloon wat die hp4d uitdrukkings vector, (pKOV410:Lip2:Exe) bevat, is bepaal deur van ‘n geoptimiseerde mRNA toebroodjie hibridisasiemetodegebruik te maak. Lotfermentasies (1.2 l) was gebruik om die produksieprofiel gedurende groei van Y. lipolytica te bepaal. Kontinüe fermentasie (1 l) is gebruik om die effek van groeitempo op die transkripsievlakke van die produk onder reguleering van die hp4d promoter te bepaal. Dit is vasgestel dat die reguleering van die sintestiese hp4d promoter onderhewig is aan die groei tempo van die organisme. Dit is bevestig deur die bepalingvan die hoeveelheid totale proteïen wat geproduseer is in 10 l voer-lotfermentasies wat teen verskillende groeitempos uitgevoer is. ‘n Sestig present toename in produksie is teweeggebring deur die organsisme teen ‘n groeitempo van 0.02 h-1 te laat groei. Dit het bevestig dat die reguleering van die hp4d promoter onderhewig is aan die groeitempo en nie die groeifase soos in literatuur genoemis nie. ‘n Strategie vir die rekombinante produksie van farmaseutiese peptiede en proteïen onder beheer van die hp4d promoter deur Y. lipolytica is dus ontwikkel. Hierdie navorsing lê die grondslag vir goedkoper rekombinante produksie van proteïen. Hierdie tegnologie kan ‘n positiewe invloed hê op gesondheid en die ekonomie van Suid Afrika deur die daarstelling van potensieel goedkoper, bekostigbare farmaseutiese medisyne. 2015-05-20T09:13:05Z 2015-05-20T09:13:05Z 2015-03 Thesis http://hdl.handle.net/10019.1/96628 en_ZA Stellenbosch University xiv, 91 pages ; illustrations application/pdf application/pdf Stellenbosch : Stellenbosch University |
| spellingShingle | Yarrowia Lipolytica Exenatide Lip2 protein production -- Economic aspects Hp42 promoter Yeast -- Fermentation UCTD Govender, Seshni Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica |
| title | Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica |
| title_full | Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica |
| title_fullStr | Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica |
| title_full_unstemmed | Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica |
| title_short | Elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by Yarrowia lipolytica |
| title_sort | elucidating the role of growth rate on the production of a fusion protein under regulation of the hp4d promoter by yarrowia lipolytica |
| topic | Yarrowia Lipolytica Exenatide Lip2 protein production -- Economic aspects Hp42 promoter Yeast -- Fermentation UCTD |
| url | http://hdl.handle.net/10019.1/96628 |
| work_keys_str_mv | AT govenderseshni elucidatingtheroleofgrowthrateontheproductionofafusionproteinunderregulationofthehp4dpromoterbyyarrowialipolytica |